Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Mitochondrial ATP generation is more proteome efficient than glycolysis.

Metabolic efficiency profoundly influences organismal fitness. Nonphotosynthetic organisms, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. Although respiration is more energy efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (that is, faster). Through quantitative flux analysis and proteomics, we find, however, that mitochondrial respiration is actually more proteome efficient than aerobic glycolysis. This is shown across yeast strains, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which promotes hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth but outgrow respiratory cells during oxygen limitation. We accordingly propose that aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Evolutionary origin and functional diversification of aminotransferases.

Aminotransferases (ATs) are pyridoxal 5'-phosphate-dependent enzymes that catalyze the transamination reactions between amino acid donor and keto acid acceptor substrates. Modern AT enzymes constitute ∼2% of all classified enzymatic activities, play central roles in nitrogen metabolism, and generate multitude of primary and secondary metabolites. ATs likely diverged into four distinct AT classes before the appearance of the last universal common ancestor and further expanded to a large and diverse enzyme family. Although the AT family underwent an extensive functional specialization, many AT enzymes retained considerable substrate promiscuity and multifunctionality because of their inherent mechanistic, structural, and functional constraints. This review summarizes the evolutionary history, diverse metabolic roles, reaction mechanisms, and structure-function relationships of the AT family enzymes, with a special emphasis on their substrate promiscuity and multifunctionality. Comprehensive characterization of AT substrate specificity is still needed to reveal their true metabolic functions in interconnecting various branches of the nitrogen metabolic network in different organisms.

Expression and characterization of spore coat CotH kinases from the cellulosomes of anaerobic fungi (Neocallimastigomycetes).

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.

Bidirectional titration of yeast gene expression using a pooled CRISPR guide RNA approach.

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae's metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.

Metabolic engineering for valorization of macroalgae biomass.

Marine macroalgae have huge potential as feedstocks for production of a wide spectrum of chemicals used in biofuels, biomaterials, and bioactive compounds. Harnessing macroalgae in these ways could promote wellbeing for people while mitigating climate change and environmental destruction linked to use of fossil fuels. Microorganisms play pivotal roles in converting macroalgae into valuable products, and metabolic engineering technologies have been developed to extend their native capabilities. This review showcases current achievements in engineering the metabolisms of various microbial chassis to convert red, green, and brown macroalgae into bioproducts. Unique features of macroalgae, such as seasonal variation in carbohydrate content and salinity, provide the next challenges to advancing macroalgae-based biorefineries. Three emerging engineering strategies are discussed here: (1) designing dynamic control of metabolic pathways, (2) engineering strains of halophilic (salt-tolerant) microbes, and (3) developing microbial consortia for conversion. This review illuminates opportunities for future research communities by elucidating current approaches to engineering microbes so they can become cell factories for the utilization of macroalgae feedstocks.

Development of platforms for functional characterization and production of phenazines using a multi-chassis approach via CRAGE.

Phenazines (Phzs), a family of chemicals with a phenazine backbone, are secondary metabolites with diverse properties such as antibacterial, anti-fungal, or anticancer activity. The core derivatives of phenazine, phenazine-1-carboxylic acid (PCA) and phenazine-1,6-dicarboxylic acid (PDC), are themselves precursors for various other derivatives. Recent advances in genome mining tools have enabled researchers to identify many biosynthetic gene clusters (BGCs) that might produce novel Phzs. To characterize the function of these BGCs efficiently, we performed modular construct assembly and subsequent multi-chassis heterologous expression using chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE allowed rapid integration of a PCA BGC into 23 diverse γ-proteobacteria species and allowed us to identify top PCA producers. We then used the top five chassis hosts to express four partially refactored PDC BGCs. A few of these platforms produced high levels of PDC. Specifically, Xenorhabdus doucetiae and Pseudomonas simiae produced PDC at a titer of 293 mg/L and 373 mg/L, respectively, in minimal media. These titers are significantly higher than those previously reported. Furthermore, selectivity toward PDC production over PCA production was improved by up to 9-fold. The results show that these strains are promising chassis for production of PCA, PDC, and their derivatives, as well as for function characterization of Phz BGCs identified via bioinformatics mining.

Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases.

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2 in biology and chemistry.

Microfabrication of a Chamber for High-Resolution, In Situ Imaging of the Whole Root for Plant-Microbe Interactions.

Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe a new 'Imaging EcoFAB' and the use of this device to image the entire root system of growing Brachypodium distachyon at high resolutions (20×, 40×) over a 3-week period. The device is capable of investigating root-microbe interactions of multimember communities. We examined nine strains of Pseudomonas simiae with different fluorescent constructs to B. distachyon and individual cells on root hairs were visible. Succession in the rhizosphere using two different strains of P. simiae was examined, where the second addition was shown to be able to establish in the root tissue. The device was suitable for imaging with different solid media at high magnification, allowing for the imaging of fungal establishment in the rhizosphere. Overall, the Imaging EcoFAB could improve our ability to investigate the spatiotemporal dynamics of the rhizosphere, including studies of fluorescently-tagged, multimember, synthetic communities.

A genetic toolbox for metabolic engineering of Issatchenkia orientalis.

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

Genome-wide identification of bacterial plant colonization genes.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica.

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.

Engineered Root Bacteria Release Plant-Available Phosphate from Phytate.

Microorganisms that release plant-available phosphate from natural soil phosphate stores may serve as biological alternatives to costly and environmentally damaging phosphate fertilizers. To explore this possibility, we engineered a collection of root bacteria to release plant-available orthophosphate from phytate, an abundant phosphate source in many soils. We identified 82 phylogenetically diverse phytase genes, refactored their sequences for optimal expression in Proteobacteria, and then synthesized and engineered them into the genomes of three root-colonizing bacteria. Liquid culture assays revealed 41 engineered strains with high levels of phytate hydrolysis. Among these, we identified 12 strains across three bacterial hosts that confer a growth advantage on the model plant Arabidopsis thaliana when phytate is the sole phosphate source. These data demonstrate that DNA synthesis approaches can be used to generate plant-associated strains with novel phosphate-solubilizing capabilities.IMPORTANCE Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems.

New voyages to explore the natural product galaxy.

Natural products are a large family of diverse and complex chemical molecules that have roles in both primary and secondary metabolism, and over 210,000 natural products have been described. Secondary metabolite natural products are of high commercial and societal value with therapeutic uses as antibiotics, antifungals, antitumor and antiparasitic products and in agriculture as products for crop protection and animal health. There is a resurgence of activity in exploring natural products for a wide range of applications, due to not only increasing antibiotic resistance, but the advent of next-generation genome sequencing and new technologies to interrogate and investigate natural product biosynthesis. Genome mining has revealed a previously undiscovered richness of biosynthetic potential in novel biosynthetic gene clusters for natural products. Complementing these computational processes are new experimental platforms that are being developed and deployed to access new natural products.

An integrated workflow for phenazine-modifying enzyme characterization.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

Computational protein design enables a novel one-carbon assimilation pathway.

We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.

A transcriptomic atlas of acute stress response to low pH in multiple Issatchenkia orientalis strains.

IMPORTANCE: Issatchenkia orientalis is a promising industrial chassis to produce biofuels and bioproducts due to its high tolerance to multiple environmental stresses such as low pH, heat, and other chemicals otherwise toxic for the most widely used microbes. Yet, little is known about specific mechanisms of such tolerance in this organism, hindering our ability to engineer this species to produce valuable biochemicals. Here, we report a comprehensive study of the mechanisms of acidic tolerance in this species via transcriptome profiling across variable pH for 12 different strains with different phenotypes. We found multiple regulatory mechanisms involved in tolerance to low pH in different strains of I. orientalis, marking potential targets for future gene editing and perturbation experiments.

Statistical 3D morphology characterization of vaterite microspheres produced by engineered Escherichia coli.

Hollow vaterite microspheres are important materials for biomedical applications such as drug delivery and regenerative medicine owing to their biocompatibility, high specific surface area, and ability to encapsulate a large number of bioactive molecules and compounds. We demonstrated that hollow vaterite microspheres are produced by an Escherichia coli strain engineered with a urease gene cluster from the ureolytic bacteria Sporosarcina pasteurii in the presence of bovine serum albumin. We characterized the 3D nanoscale morphology of five biogenic hollow vaterite microspheres using 3D high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) tomography. Using automated high-throughput HAADF-STEM imaging across several sample tilt orientations, we show that the microspheres evolved from a smaller more ellipsoidal shape to a larger more spherical shape while the internal hollow core increased in size and remained relatively spherical, indicating that the microspheres produced by this engineered strain likely do not contain the bacteria. The statistical 3D morphology information demonstrates the potential for using biogenic calcium carbonate mineralization to produce hollow vaterite microspheres with controlled morphologies. STATEMENT OF SIGNIFICANCE: The nanoscale 3D structures of biomaterials determine their physical, chemical, and biological properties, however significant efforts are required to obtain a statistical understanding of the internal 3D morphology of materials without damaging the structures. In this study, we developed a non-destructive, automated technique that allows us to understand the nanoscale 3D morphology of many unique hollow vaterite microspheres beyond the spectroscopy methods that lack local information and microscopy methods that cannot interrogate the full 3D structure. The method allowed us to quantitatively correlate the external diameters and aspect ratios of vaterite microspheres with their hollow internal structures at the nanoscale. This work demonstrates the opportunity to use automated transmission electron microscopy to characterize nanoscale 3D morphologies of many biomaterials and validate the chemical and biological functionality of these materials.